Double phosphorylation‐induced structural changes in the signal‐receiving domain of IκBα in complex with NF‐κB

Abstract

Activation of the transcription factor NF-κB requires degradation of its physiological inhibitor IκBα in order to allow nuclear translocation of NF-κB. NF-κB activity links inflammation and carcinogenesis and makes its signaling pathway an important target for therapeutic intervention. The signal-receiving N-terminal domain (SRD) of the NF-κB inhibitor IκBα harbors the sites of post-translational modifications (Ser32 and 36) directed by the IκB kinase (IKK) complex. The SRD was originally recognized to be highly disordered, but was recently shown to possess stable secondary structural elements. Identifying and characterizing the structural effects that arise from phosphorylation may explain how phosphorylation regulates the IκBα-NF-κB protein complex. Therefore, the effect of post-translational mono- and double-phosphorylation of the serine residues of the SRD was analyzed. The structural modifications of the IκBα-NF-κB protein-protein complex due to mono-phosphorylation of either Ser32 or Ser36 amino acid residues or simultaneous phosphorylation were investigated by means of molecular dynamics simulations. Mono-phosphorylation at either Ser32 or Ser36 was not sufficient to induce significant structural changes in the secondary structure of the SRD of IκBα. Double-phosphorylation yielded an increase in distance between the Cα atoms of these serine residues, indicative of a structural change. Only this two-fold phosphorylation induced the extended, more stabilized conformation of the degron motif which renders it accessible by the E3 ligase. In summary, these results provide insight into the conformational changes induced in IκBα proteins upon phosphorylation that are vital to their signaling dynamics and enable us to propose a model for the phosphorylation of the SRD. Proteins 2016; 85:17-29. © 2016 Wiley Periodicals, Inc.

Post-translational Modifications of IκBα: The State of the Art

“…The IKK complex can be activated by lipopolysaccharides (LPSs), viral proteins, oxygen free radicals, cytokines and other stimuli; it then phosphorylates the Ser36 and Ser32 residues in the SRD region of the N-terminus of IκBα (Traenckner et al, 1995;Hayden and Ghosh, 2004;Kato et al, 2012;Ko et al, 2017;Liu et al, 2018;Safi et al, 2018), leading to ubiquitination and the subsequent degradation of the IκBα in proteasome (Figure 2). IKK-dependent IκBα phosphorylation at the serine residues occurring in the cytosol is a key step in the release of active NF-κB and its nuclear translocation (Yazdi et al, 2017). A molecular dynamics simulation experiment showed that IKK first phosphorylates IκBα at Ser36, changing the local conformation of the N-terminal region and increasing the relative solvent-accessible surface area of Ser32.…”

“…A molecular dynamics simulation experiment showed that IKK first phosphorylates IκBα at Ser36, changing the local conformation of the N-terminal region and increasing the relative solvent-accessible surface area of Ser32. This enables Ser32 to interact with IKK via phosphorylation (Yazdi et al, 2017).…”

“…Then, the Met1 and Gln3 residues in the N-terminal move away from p-Ser32 to p-Ser36. Finally, the amides in the main chain of Met1 interact with the phosphate group of p-Ser36, whereas those in the main and side chains of Gln3 combine with the backbone carbonyl group of p-Ser36 to form hydrogen bonds (Yazdi et al, 2017). These changes result in the stabilization of the N-terminal tail.…”

Targeting the NF-κB/IκBα complex via fragment-based E-Pharmacophore virtual screening and binary QSAR models

“…However, it was found that phosphorylation of the PEST sequence is not critical for the binding affinity, but only slightly increase the association rate between NF κB and IκBα by perhaps enhancing the folding mechanism of binding 64 . Casein kinase II is known to phosphorylate residues Ser283, Ser288, Thr291, Ser293, and Ser299 64 .…”

Model-based identification of TNFα-induced IKKβ-mediated and IκBα-mediated regulation of NFκB signal transduction as a tool to quantify the impact of drug-induced liver injury compounds

“…IκBα in complex with NFκB is a substrate for the IKK complex. 23 , 24 Phosphorylation of IκBα prompts it for ubiquitination leading eventually to the degradation of IκBα and the release and nuclear translocation of NFκB. These steps provide another important regulatory mechanism to control the activation of NFκB signal transduction downstream of the TNFR complex.…”